Role of RelA-synthesized (p)ppGpp and ROS-induced mutagenesis in de novo acquisition of antibiotic resistance in E. coli

Summary The stringent response of bacteria to starvation and stress also fulfills a role in addressing the threat of antibiotics. Within this stringent response, (p)ppGpp, synthesized by RelA or SpoT, functions as a global alarmone. However, the effect of this (p)ppGpp on resistance development is poorly understood. Here, we show that knockout of relA or rpoS curtails resistance development against bactericidal antibiotics. The emergence of mutated genes associated with starvation and (p)ppGpp, among others, indicates the activation of stringent responses. The growth rate is decreased in ΔrelA-resistant strains due to the reduced ability to synthesize (p)ppGpp and the persistence of deacylated tRNA impeding protein synthesis. Sluggish cellular activity causes decreased production of reactive oxygen species (ROS), thereby reducing oxidative damage, leading to weakened DNA mismatch repair, potentially reducing the generation of mutations. These findings offer new targets for mitigating antibiotic resistance development, potentially achieved through inhibiting (p)ppGpp or ROS synthesis.


INTRODUCTION
The spectrum of bacterial defense mechanisms against antimicrobials encompasses, but is not confined to, target alterations, upregulation of efflux pumps, reduction of cellular permeability, and modification of antibiotics. 1 Such complicated mechanisms necessitate specific physiological activity within the cells.Stress caused by exposure to antibiotics and the subsequent increased protein synthesis triggers in bacteria the same stringent response as during nutrient starvation.This response is mediated by the synthesis of the signaling nucleotides guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), collectively termed (p)ppGpp. 2,3Bacterial resistance to antibiotics is caused by cellular adaptation in combination with genomic DNA mutations or acquisition of exogenous DNA. 4 There is evidence indicating that molecular alterations seen as a result of the stringent response are related to the development of resistance mutations in both ways. 5he enzyme GDP/GTP pyrophosphokinase RelA plays a crucial role in the stringent response by catalyzing the synthesis of (p)ppGpp. 6ence it is hypothesized that the knockout of relA results in the suppression of (p)ppGpp synthesis.Deletion of relA in E. coli results in reduced mutation rates in multiple amino acid auxotrophic strains, and a direct correlation has been established between the concentration of (p) ppGpp and the mutation rate. 7,8Furthermore, the involvement of (p)ppGpp extends to the regulatory control of class 1 integron integrases which enable bacteria to express and capture antibiotic resistance gene cassettes under starvation-induced stringent response in biofilms. 9he regulatory impact of (p)ppGpp, accomplished through its direct interaction with RNA polymerase, manifests itself in the modulation of transcription initiation at specific gene promoters.Additionally, (p)ppGpp is involved in modulating the production and activity of the RNA polymerase sigma factor RpoS, which serves as the master transcriptional regulator of the general stress response. 10HipA and HipB are components of a type II toxin-antitoxin (TA) system.It has been demonstrated that HipA expression activates ppGpp synthesis mediated by RelA. 11On the other hand, HipB functions as an antitoxin, counteracting the toxic effects of the cognate toxin HipA. 12Consequently, the knockout of hipB may potentially result in an elevated level of ppGpp.This study addresses the role of stringent response mediated stress responses on antibiotic resistance development upon long-term exposure to sub-lethal levels of antibiotics, by means of evolution experiments on four (p)ppGpp associated E. coli knockout strains DrelA, DrpoS, DhipA, and DhipB.
Reactive oxygen species (ROS) produced upon exposure to sublethal levels of bactericidal antibiotics affect development of resistance according to a hormesis mechanism. 13,14That is, high levels of ROS kill cells, while sub-lethal levels of ROS affect cellular DNA inducing mutations that may be beneficial in promoting the formation of antibiotic resistance. 15Besides their specific antibiotic-target interactions, bactericidal antibiotics, such as b-lactams, quinolones, and aminoglycosides, stimulate oxidation of NADH via the electron transport chain. 16As a consequence, superoxide formation is also enhanced, which in turn promotes hydroxyl radical formation via the Fenton reaction. 17,18These byproducts, collectively called ROS, are very reactive with cellular components, especially DNA, for example by oxidizing guanine to 8-hydroxy-2 0 -deoxyguanosine (8-HOdG), which may increase mutation rates. 19The oxidative response induced by ROS and the stringent response triggered by (p)ppGpp are both metabolic feedback loops by which cells respond to various stresses.The connection between these two stress responses and their implications for acquisition of resistance under antibiotic exposure remain to be elucidated.
In this investigation, we quantified the ROS levels during antibiotic resistance development within (p)ppGpp-synthesis mutant strains, simultaneously assessing the inflicted damage due to ROS and the subsequent repair processes.We propose a mechanism for the interplay between the stringent stress response and oxidative stress responses and corroborate the association between ROS-mediated hormesis and the evolution of antimicrobial resistance, particularly at relatively moderate ROS levels.

Lower rates of resistance development in relA or rpoS knockout strains
To investigate the effect of (p)ppGpp on the acquisition of de novo antibiotic resistance, we exposed fully susceptible E. coli wild-type and four single-gene knockout strains DrelA, DrpoS, DhipA, and DhipB to stepwise-increasing sub-lethal concentrations of four antibiotics (Figure 1).The resistance evolution experiments started at one-quarter of the minimum inhibitory concentration (MIC) for amoxicillin (0.5 mg/mL), enrofloxacin (0.125 mg/mL), kanamycin (2 mg/mL), and tetracycline (0.5 mg/mL).
During the early stages of exposure to amoxicillin, the resistance acquisition rate of the DrelA was lower than that of the WT strains (Figure 1A).At day 10 the MIC of the DrelA strain made resistant to amoxicillin was significantly lower than that of the WT (Figure 1Q).The final resistance concentrations of the DrelA were in two independent experiments 512 mg/mL and 1,024 mg/mL, respectively, versus 2,048 mg/mL for the wild-type strains (Figure 1A).Similar to the DrelA, the MIC of the DrpoS at day 30 was significantly lower than that of the wild-type strains (Figure 1Q).The maximum resistance concentration of one replicate of the DrpoS was 256 mg/mL, and both replicates reached their final resistance concentrations later than the WT-resistant strain (Figure 1E).There were no apparent differences observed in the development of amoxicillin resistance between the wild-type and DhipA strains (Figure 1I).Resistance development of DhipB progressed at a higher rate than that of the wild-type strains during the middle stages, but the final level equaled that of the wild-type (Figure 1M).The MIC of the DhipB at day 20 was higher than that of the WT strain (Figure 1Q).
During enrofloxacin exposure, clear differences between the strains could be observed after day 10 (Figures 1B-1F, 1J, and 1N).At day 20, the MIC of the DrelA-resistant strain and DrpoS-resistant strain were significantly lower than the WT-resistant strain, and all these strains exhibited lower MIC values compared to the DhipB-resistant strain (Figure 1R).The final resistance concentrations of DrelA and DrpoS were 8 mg/mL and 128 mg/mL, respectively, considerably lower than the wild-type strains' concentration of 1,024 mg/mL (Figures 1B and 1F).One replicate of the DhipA ceased to grow at day 13, but the other one exhibited a similar resistance acquisition rate and MIC as the wild-type strains (Figures 1J and 1R).Similarly, resistance of one replicate of the DhipB stopped increasing at day 20 of 64 mg/mL.However, the other replicate showed a higher resistance acquisition rate than the wild-type strains, and its final resistance concentrations were double those of the WT-resistant strains (Figures 1N and 1R).
During kanamycin exposure, the rate of resistance acquisition and the MIC at days 10 and 20 were relatively consistent across all strains (Figures 1C-1G, 1K, 1O, and 1S).However, the maximum resistance concentrations of DrelA and DrpoS were both 1,024 mg/mL, which was half that observed in the WT-resistant strains (Figures 1C and 1G).The hipA knockout had no meaningful effect on the resistance development (Figure 1K).Remarkably, the DhipB reached kanamycin resistance concentrations of 2,048 mg/mL at day 20, which was faster than the wild-type strains (Figure 1O).
In the case of the bacteriostatic antibiotic tetracycline, the strains reached their final resistance concentrations at 32 mg/mL, which was low compared to the bactericidal antibiotics (Figures 1D-1H, 1L, and 1P).Additionally, the resistance acquisition rate, and MIC at day 10 or day 20 were all roughly the same in each strain (Figure 1T).

Mutations in stress response genes accompany antibiotic resistance
At the end of the antibiotic resistance evolution experiments, the genomic DNA of the final resistant strains was sequenced entirely to identify mutations that may influence development of resistance.The mutations that accompanied antibiotic resistance were clustered into different groups according to their functions as defined by the Comprehensive Antibiotic Resistance Database and UniProt (Tables 1, 2, 3, 4, and 5). 20,21he mutations that relate to metabolism or that are functionally unknown are given in the appendix (Table S1).
Amoxicillin-resistant strains displayed shared mutations within the ampC promoter region (Table 1).These mutations likely contribute to an upregulation in the levels of the b-lactamase AmpC. 22,235][26] Mutations in envZ and ompC, which encode the sensor histidine kinase EnvZ (OmpB) and outer membrane porin OmpC, respectively, were frequently observed. 27These mutations may result in reduced antibiotic entry into the cells. 28Occasional mutations were also observed in genes associated with efflux pumps or target alterations, albeit less frequently.Notably, a chpS deletion mutation was identified in one replicate of the DrelA strain, and an upstream mutation in the dps gene occurred in another replicate.The antitoxin coding gene chpS together with the toxin gene chpB belong to a type II toxin-antitoxin (TA) system, potentially involved in the regulation of cell growth. 29dps codes for a DNA protection protein for starvation response, it protects DNA against multiple stresses, including oxidative stress. 30Among the DhipA_2-resistant strain, three missense variations were found in the toxin coding gene ghoT.Together with the antitoxin gene ghoS, this type V TA system plays a role in limiting cell growth during antibacterial stress. 31In the DhipB_2-resistant strain, a mutation was observed in the hfq gene, which encodes an RNA-binding protein.Hfq is part of a gene network that facilitates stress-induced mutagenesis (SIM) in E. coli. 32nrofloxacin targets DNA gyrase and topoisomerase IV thereby inhibiting bacterial DNA synthesis. 33The shared mutations occurring in the DNA gyrase subunit A coding gene, gyrA (Table 3), and located on the quinolone resistance-determining region (QRDR), result in reduced affinity for quinolones. 34Resistant strains of the WT, DhipA_1, and DhipB_2 acquired mutations in parC, gyrB, or parE, enhancing their resistance and enabling them to reach higher concentrations of enrofloxacin resistance compared to other strains (Figures 1J and 1N).Another resistance mechanism these strains evolved operated through efflux pumps that expel the antibiotic.The main mutated gene associated with this mechanism was acrR, which encodes the HTH-type transcriptional regulator AcrR, known as a repressor of the AcrAB-TolC multidrug efflux complex. 35In the WT-resistant strain, DNA repair-related DNA helicase coding genes dinG and yoaA were mutated. 36,37Interestingly, the (p)ppGpp synthase/hydrolase coding gene spoT exhibited a mutation in the DrelA_2, possibly activating the stringent response in these cells. 38The dps mutation found in the amoxicillin-resistant DrelA_2 appeared again but in a different position in the enrofloxacin-resistant strain DrpoS_1.In the DhipA_1 strain, a mutation occurred in the cell division inhibitor SulA, a component of the SOS system. 39Furthermore, two mutations were identified in the hydrogen peroxide-inducible gene activator coding gene oxyR in the DhipB_2 resistant strain, suggesting that the strain is under oxidative stress caused by ROS. 40he gene mutated in each of the kanamycin-resistant strains was fusA, with the predominant mutation being T393I (Table 4).The fusA gene encodes the elongation factor G, which plays a crucial role in ribosomal translocation during translation elongation. 41These mutations in fusA may contribute to the reduced binding efficiency of kanamycin.Another frequently mutated gene, sbmA, encodes an innermembrane transport protein that in mutated form has been proven to impede the uptake of antimicrobial peptides. 42,43While the function remains unknown, mutations in the oligopeptide transport protein genes oppB, oppD, or oppF have been implicated in kanamycin resistance. 44,45Interestingly, a mutation in the bifunctional (p)ppGpp synthase/hydrolase spoT gene was also identified in the kanamycin-resistant strain DrelA_2.
There was no shared mutated gene observed among all tetracycline-resistant strains (Table 5).However, a V57L mutation in the ribosomal subunit coding gene rpsJ was observed in the WT, DrelA_2, DhipB_1, and DhipB_2 strains.This mutation may protect the antibiotic target.Furthermore, a 44_45del mutation within the outer membrane lipoprotein coding gene mlaA was observed in the DrelA and DhipA resistant strains.A G315S mutation in the spoT gene that codes for a key enzyme of the stringent response was observed both in the kanamycin-resistant strain DrelA_2 and in the tetracycline-resistant DrelA strains (Table 4).The tetracycline-resistant strains shared several common mutated genes compared to other antibiotic-resistant strains.Mutations in the envZ gene and the RNA polymerase subunit coding genes rpoBCD were observed under both amoxicillin and tetracycline treatment.These mutations may cause reduced antibiotic permeability, and alterations in antibiotic targets, respectively (Table 1).Mutations were also detected in the efflux pump regulator gene acrR and the multidrug efflux pump subunit coding gene acrB under tetracycline exposure, while an acrA mutation was identified during enrofloxacin treatment (Table 3).These findings suggest that these mutations may not be specific to a particular antibiotic.
Reduced oxidative stress and DNA damage in DrelA-resistant strains during exposure to bactericidal antibiotics In these de novo antibiotic resistant strains, mutations were observed not only in genes directly associated with drug resistance but also in a variety of genes involved in regulating cellular stress responses (Tables 1, 2, 3, 4, and 5).7][48] To further investigate that notion, we performed RNA differential quantification on the DrelA-resistant strains that exhibited a slower evolution rate compared to the WT-resistant strains during bactericidal antibiotic treatment.(Figures 1A-1D).The differentially expressed genes associated with these stress responses were categorized according to the Gene Ontology Biological Process (GO-BP), with a  In the table, the size of the fragment, the amplification factor, and upstream and downstream of the genes contained in the fragment are listed for each strain tested.
log 2 fold change cutoff of 2 applied to select the relevant genes (Figure 2A).Notably, a substantial proportion of the differentially expressed genes were related to the response to oxidative stress and DNA damage (Figure 2A).Therefore, we measured the ROS production levels and oxidation-mediated DNA damage characteristic 8-HOdG level in these strains (Figures 2B and 2C).Among all the DrelA and WT final resistant strains, the ROS production levels in cells exposed to bactericidal antibiotics were higher than in cells exposed to the bacteriostatic antibiotic tetracycline (Figure 2B).In addition, significantly lower ROS production levels were detected in the DrelA-resistant strains compared to WT-resistant strains under exposure to bactericidal antibiotics amoxicillin, enrofloxacin, and kanamycin (Figure 2B).Moreover, the 8-HOdG production level was significantly increased in the WT-resistant strains exposed to the maximum concentrations of bactericidal antibiotics compared to the untreated naive cells (Figure 2C).In the DrelA-resistant strains, enrofloxacin or kanamycin treatment caused clearly higher 8-HOdG production levels.Similar to the ROS production level, the 8-HOdG production level in DrelA-resistant strains was significantly lower than in WT-resistant strains under bactericidal antibiotics treatment.
We assumed that ROS levels decreased as a result of the growth rate reduction caused by the knockout of the (p)ppGpp synthesis gene, relA.This lower growth rate would lead to a decline in respiratory chain activity, subsequently resulting in reduced ROS production.To verify this hypothesis, we measured the growth rates of the WT and DrelA final resistant strains under maximum antibiotic concentrations (Figure 2D).There was no difference in growth rates between the WT and DrelA naive strains without antibiotic in the medium.However, the growth rate of the DrelA-resistant strains was significantly decreased compared to that of the WT-resistant strains when exposed to each antibiotic.In summary, our study findings indicate that the DrelA-resistant strains exhibit reduced DNA damage caused by ROS during exposure to bactericidal antibiotics, primarily due to the decreased growth rate induced by the relA knockout.

Knockout of relA resulted in decreased transcription levels of DNA repair genes during exposure to bactericidal antibiotics
In response to DNA damage, cells activate various mechanisms to detect and repair that damage.To identify the differentially expressed genes involved in DNA damage repair, we focused on the WT and DrelA-resistant strains under maximum concentrations of antibiotics (Figure 2A).The DrelA mutant was chosen for comparison with the WT as it lacks a key enzyme of the stringent response.Genes associated with DNA repair with a log 2 fold change greater than 2 compared to untreated naive cells were selected and are represented in a heatmap (Figure 3).
During amoxicillin treatment, both the WT and DrelA-resistant strains exhibited upregulation of sulA and recA, with similar expression levels.Transcription levels of lexA, recN, umuC, umuD, mutM, nudG, yebG, and tisB were upregulated in the WT but attenuated in the DrelA-resistant strain.Enrofloxacin treatment induced the highest number of differentially expressed genes, including sulA, recANX, umuCD, dinBDI, uvrA, yebG, tisB, symE, and yafNOP, and caused the highest upregulation levels in both the WT and DrelA-resistant strains.The transcription levels of recF, holD, mutM, vsr, and ycaQ were lower in the DrelA-resistant strain, while lexA exhibited higher expression compared to the WT during exposure to enrofloxacin.Kanamycin treatment resulted in minor differences between the WT and DrelA-resistant strains.Even so, the transcription level of lexA, sulA, recN, umuCD, and yebG were higher in the WT.Under tetracycline treatment, recF exhibited higher upregulation level in the WT compared to DrelA, whereas in yafNOP displayed the opposite trend.Other genes showed relatively similar expression levels between the two tetracycline-resistant strains.

DISCUSSION
In this experimental design, the single gene knockout of relA or rpoS decelerated the acquisition of resistance upon exposure to bactericidal antibiotics.This slowdown may be attributed to the regulatory function of (p)ppGpp and the mutagenic effect of ROS (Figure 4).Under normal conditions without antibiotic exposure, the growth rate of the naive wild-type and DrelA cells was roughly the same (Figure 2D).Bacteria can intrinsically synthesize various amino acids even in a minimal medium, ensuring sufficient aminoacylated tRNAs for translation elongation and maintaining normal growth and reproductive functions (Figure 4A).Bacteria employ multiple strategies to respond to antibiotics, including the synthesis of enzymes to degrade antibiotics, reducing drug entry, increasing drug efflux, and enhancing target modification. 49This increased synthesis of corresponding proteins leads to higher levels of emerging deacylated tRNA.Binding of deacylated tRNAs to the A site of the ribosome leads to an interruption of the translation elongation process. 50However, RelA can recognize deacylated tRNAs bound to ribosomes and then synthesize (p)ppGpp, temporarily redirecting transcription from growth-related genes to genes involved in stress resistance and starvation survival. 51,52In addition, (p)ppGpp can also increase the amount of aminoacylated tRNAs through amino acid synthesis and proteolysis, thereby ensuring an effective bacterial antibiotic stress response. 53During exposure to bactericidal antibiotics, the drugtarget interactions stimulate the acceleration of the electron transport chain, resulting in the formation of by-product ROS. 17,54ROS-induced DNA damage and cell repair will elevate the mutation rate, thereby creating a larger window of opportunity for beneficial mutations to arise, thus accelerating the formation of antimicrobial resistance during prolonged antibiotic exposure 13,15,46 (Figure 4B).When the relA gene is knocked out, the absence of RelA-induced (p)ppGpp synthesis can lead to the persistence of deacylated tRNAs. 55Due to the hysteresis intrinsic synthesis of amino acids in minimal media, this reduces the cell's ability to respond effectively to antibiotics, impairs its capacity to sustain growth, and weakens activity of the electron transport chain.Consequently, ROS production falls below a tipping point that is beneficial for increased non-lethal mutation rates, thus hindering drug resistance formation (Figure 4C).As a global regulator, (p)ppGpp interacts with the DnaK repressor (DksA) by directly binding RNA polymerase and influencing the transcription of specific genes. 56In addition, (p)ppGpp modulates several sigma factors, such as the RNA polymerase sigma factor RpoS, which guides RNA polymerase transcription in response to specific stress conditions. 52RpoS serves as the master transcriptional regulator of the general stress response, being involved not only in starvation response but also in various other responses, including pH changes, oxidative stress, high temperature, and osmotic pressure. 57Importantly, RpoS promotes the generation of SIM, enabling cells to improve their environmental adaptability through evolution. 58Upon exposure to antibiotics, RpoS enhances the transcription of low-fidelity DNA polymerases in response to DNA damage. 59This increases the mutation rate during the repair process, potentially contributing to the formation of antimicrobial resistance. 46,47The resistance evolutions of the rpoS knockout strain were slower than that of the wild type, which aligns with the aforementioned theory (Figures 1E-1H).HipA and HipB belong to the type II TA system. 11HipA can phosphorylate the glutamate (Glu) tRNA ligase (GltX), which results in the accumulation of uncharged tRNA (Glu), subsequently triggering the synthesis of (p)pp(G)pp by RelA. 60HipB serves as a transcriptional repressor that counteracts the actions of HipA, thus inhibiting the formation of high-persister cells induced by HipA in response to antibiotics. 12,61The resistance acquisition rates of the wild-type and DhipA strains exhibited similar patterns (Figures 1I-1L).However, the resistance evolution rate of the DhipB strains was faster than that of the wild type upon exposure to bactericidal antibiotics (Figures 1M-1P).These observations suggest that the deletion of the hipA gene does not affect the acquisition of resistance, whereas the absence of hipB weakens the neutralization of HipA, and the subsequently induced (p)ppGpp appears to accelerate the development of antibiotic resistance.Upon phosphorylation of GltX by HipA, the accumulation of uncharged tRNAs leads to the upregulation of overall amino acid synthesis induced by (p)ppGpp, thereby enhancing the cells' tolerance to antibiotics.This contrasts with the deceleration of resistance acquisition observed in the DrelA strains, yet ultimately underscores the correlation between (p)ppGpp and resistance acquisition.
Resistant cells exhibited common target-specific mutations in response to the three bactericidal antibiotics. 13,26,44For instance, mutations in the ampC gene were observed under amoxicillin treatment, gyrA gene mutations occurred under enrofloxacin treatment, and fusA gene mutations were detected under kanamycin treatment (Tables 1, 2, 3, and 4).Additionally, non-target-specific mutations in genes related to efflux pumps and antibiotic permeability reduction were also involved in the development of antibiotic resistance.Furthermore, certain genetic mutations not directly associated with antibiotic resistance drew our attention.For example, in DrelA-resistant strains, frequent mutations in the spoT gene strongly suggested that these strains were under stringent stress.These spoT gene mutations may serve as compensatory evolution to regulate the synthesis of (p)ppGpp. 62Moreover, dps mutations appeared in both DrelA and DrpoS resistant strains following amoxicillin or enrofloxacin exposure (Tables 1 and 3).4][65] However, this in turn reduces the mutagenic responses by ROS-induced DNA damage repair.In contrast, the SIM coding gene hfq, SOS system coding gene sulA, and oxidative stress inducible gene oxyR found in DhipA and DhipB resistant strains predicted the emergence of damage-repairinducing mutations.
The role of ROS as a secondary killing mechanism of bactericidal antibiotics has been elucidated in recent years. 17,19,66ROS-induced stress is involved in de novo antimicrobial resistance acquisition according to the principle of hormesis. 67,68Knockout of ROS-removing genes accelerated resistance development, whereas the ROS scavenger thiourea attenuated it. 13Similarly, in this study, we observed that the knockout of relA resulted in reduced ROS production in resistant strains, leading to decreased production of the signaling molecule 8-HOdG associated with ROS-induced damage (Figures 2B and 2C).As one of the possible oxidative base damages, 8-HOdG arises from guanine hydroxylation at the nucleotide pool or genomic DNA level. 69It induces guanine-to-thymine mutations during replication as it prefers to pair with adenine instead of cytosine. 70,71Bacteria engage several DNA repair genes through the SOS stress system to respond to the ROS-caused DNA damage.ROS-induced DNA oxidative damage is primarily repaired through the DNA base excision repair (BER) pathway. 72Formamidopyrimidine-DNA glycosylase MutM is involved in the BER of DNA damaged by oxidation, it recognizes lesions such as 8-HOdG or thymine glycol and removes them. 73We found that the transcription levels of mutM were lower in DrelA-resistant strains compared to WT-resistant strains after exposure to amoxicillin or enrofloxacin (Figure 3).Additionally, the error-prone DNA polymerase coding genes umuCD exhibited a lower transcription level in DrelA-resistant strains after exposure to amoxicillin or kanamycin.This low-fidelity polymerase V lacks intrinsic 3 0 -5 0 exonuclease proofreading activity, and thus easily induces mutations during DNA repair. 74This appears to act as a protective mechanism by increasing the generation of resistance-associated mutations, which were reduced after the knockout of relA.Changes in the expression levels of genes related to DNA repair were found upon exposure to non-DNA-specific targeting antibiotics, such as amoxicillin and kanamycin.This observation further underscores that a shared mutagenic pathway is attributable to ROS.

Limitations of the study
Our research connects the stringent stress response together with the oxidative stress response, suggesting, but not decisively proving that RelA-synthesized (p)ppGpp plays a crucial role in antimicrobial resistance development by regulating bacterial growth rate and ROS formation.This study shows that although high levels of ROS are lethal, moderate levels of ROS enhance the rate of resistance acquisition by increasing damage-induced mutagenesis.Therefore, the principle of hormesis applies: high levels of stress caused by exposure to high concentrations of antibiotics are lethal, but low-level exposure induces resistance mutations, making the cell resistant, which is beneficial.The intracellular mechanism is inactive when the synthesis of (p)ppGpp is limited, the production of by-product ROS is reduced, ultimately slowing down the development of antibiotic resistance.These results suggest potential strategies to reduce resistance development, such as the The right column provides information on the regulated genes and their corresponding functions.design of (p)ppGpp inhibitor relacin and its analogs. 75,76However, the specific ROS level that reduces the development of resistance needs to be further elucidated.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

REAGENT or RESOURCE SOURCE IDENTIFIER
Bacterial and virus strains The antibiotic-sensitive single-gene-knockout E. coli K12 strains DrelA, DrpoS, DhipA, and DhipB, and the wild-type MG1655 were cultured in a phosphate buffered (100 mM NaH 2 PO 4 $ 2H 2 O) defined minimal Evans medium supplemented with 55mM glucose (pH 6.9). 86The kanamycin resistance cassette in knockout strains was substituted with temperature-sensitive pCP20 plasmid. 77The cultures were incubated in 10 mL tubes at a temperature of 37 C and constantly shaken at 200 rpm.

METHOD DETAILS Evolution experiments and MIC determination
De novo resistance acquisition evolution experiments were executed following established protocols. 87In brief, a single clone of each strain was cultured in tube contained Evans medium overnight, and an appropriate volume of each culture was inoculated into a fresh medium resulting in an initial optical density (OD) at 600 nm of 0.1.Antibiotics were added to the respective cultures at a concentration of one-fourth of the MIC and incubated overnight.A control group without antibiotics was also maintained.If, on a subsequent day, the OD 600 of the antibiotic-treated culture exceeded 75% of the antibiotic-free culture, a portion of this culture was transferred to a fresh medium at an OD 600 of 0.1.The antibiotic concentration was then doubled and maintained in two separate tubes.On the third day, if the OD 600 of the high-concentration antibiotic-treated culture surpassed 75% of the low-concentration antibiotic culture, a portion of the high-concentration culture was transferred to a fresh medium.Otherwise, the low-concentration antibiotic culture was chosen.This process continued with an incremental doubling of the antibiotic concentration until stable resistant strains were established.Cultures without antibiotics were continuously incubated daily throughout the evolution experiment as a control.Each strain's evolution experiment was independently replicated at least twice.MIC testing was performed three times a week using a spectrophotometer plate reader (Thermo Fisher Scientific) to monitor resistance development.Cultures with an initial OD 600 of 0.05 were incubated in 150 mL of medium within a 96-well plate.Antibiotic concentrations ranged from 0.5 to 2048 mg/mL, with two-fold increment steps.Following overnight incubation, the MIC was defined as the lowest concentration with a final OD 600 below 0.2.MIC values at day 10, day 20, and day 30 for each strain against antibiotics were documented during the evolution experiments.Each biological replicate contains three technical replicates.The data were presented as means G SD, statistical significance was determined using a one-way ANOVA, *p < 0.05, **p < 0.01.

Figure 1 .
Figure 1.Effect of relA, rpoS, hipA, and hipB knockouts on antibiotics resistance development (A-P) Antibiotic resistance development was evaluated in E. coli wild-type MG1655 (WT) and single gene knockout strains DrelA, DrpoS, DhipA, and DhipB against amoxicillin (A, E, I, and M), enrofloxacin (B, F, J, and N), kanamycin (C, G, K, and O), and tetracycline (D, H, L, and P).The x axis represents the duration of evolution in days, while the y axis represents the concentration of acquired resistance.(Q-T) Comparison of minimum inhibitory concentration (MIC) at day 10, day 20, and day 30 for each strain (WT, DrelA, DrpoS, DhipA, and DhipB) against amoxicillin (Q), enrofloxacin (R), kanamycin (S), and tetracycline (T) during the process of antibiotic resistance acquisition.Data are presented as means G SD, statistical significance was determined using a one-way ANOVA, n R 3, *p < 0.05, **p < 0.01.

Figure 2 .
Figure 2. Differential oxidative stress and DNA damage in DrelA resistant strains during bactericidal antibiotics exposure (A) Transcriptomic analysis of E. coli resistant WT and DrelA strains after antibiotics exposure, revealing the relative abundance of regulated genes.Functional cluster according to the Gene Ontology Biological Process (GO-BP) and based on log 2 fold change values higher than 2 and lower than À2.The selected clusters refer to whole genome sequencing results and are depicted in the scale bar.The abundance shows the percentage of genes within each selected cluster relative to the total number of upregulated and downregulated genes with a log 2 fold change cutoff higher than 2. ROS, reactive oxygen species; TCA, citric acid cycle; ETC, electron transport chain; AMO, amoxicillin-treated; ENR, enrofloxacin-treated; KAN, kanamycin-treated; TET, tetracycline-treated. (B) Assessment of ROS production levels in resistant E. coli WT and DrelA strains following exposure to maximum concentrations of each antibiotic.The Y axis represents the percentage of ROS-producing cells within each population.Data are presented as means G SD. Statistical significance was determined using a one-way ANOVA, N = 3, *p < 0.05, **p < 0.01, ***p < 0.001.(C) Evaluation of 8-HOdG production levels in resistant E. coli WT and DrelA strains after treatment with maximum antibiotic concentrations.The Y axis represents the 8-HOdG concentrations divided to DNA concentrations.Data are presented as means G SD. Statistical significance was determined using a one-way ANOVA, N = 3, *p < 0.05, **p < 0.01, ***p < 0.001.CON, untreated naive cells.(D) Growth rate of resistant E. coli WT and DrelA strains after treatment with maximum antibiotic concentrations.Data are presented as means G SD. Statistical significance was determined using a one-way ANOVA, N = 3, *p < 0.05, **p < 0.01, ns, not significant.CON, naive cells without antibiotics.

Figure 3 .
Figure 3. Gene transcription levels of DNA damage-repair-associated genes The heatmap represents the log 2 fold change values of gene expression, with blue color indicating upregulated genes and yellow color indicating downregulated genes.Genes associated with DNA damage-repair were selected based on a log 2 fold change cutoff greater than 2 in any treatment group.The right column provides information on the regulated genes and their corresponding functions.

Figure 4 .
Figure 4. Model depicting the role of RelA synthesized (p)ppGpp on de novo acquisition of antibiotic resistance (A) Aminoacylated tRNA is provided to synthesize proteins during translation elongation to maintain normal growth and reproduction.(B) In response to antibiotics, cells synthesize additional proteins, which leads to insufficient aminoacylated tRNAs.Deacylated tRNAs binding to ribosome triggers (p)ppGpp to regulate amino acid synthesis, thereby reacting to antibiotics.Bactericidal antibiotics and the cell target interactions generate sublethal levels of ROS as a by-product through the accelerated operation of the ETC.Mutagenesis by ROS contributes to the development of drug resistance.(C) Knockout of relA results in sustained amino acid starvation.Cellular responses to antibiotics and production of ROS will be reduced.The development of drug resistance is slowed down.

Table 1 .
Mutations associated with resistance development after amoxicillin exposure OMP, outer membrane protein; SIM, stress-induced mutagenesis.a Upstream mutation.

Table 3 .
Mutations associated with resistance development after enrofloxacin exposure a Upstream mutation.

Table 2 .
Copy numbers of gene amplification regions including ampC after amoxicillin exposure

Table 4 .
Mutations associated with resistance development after kanamycin exposure

Table 5 .
Mutations associated with resistance development after tetracycline exposure a Upstream mutation.

TABLE
d RESOURCE AVAILABILITY B Lead contact B Materials availability B Data and code availability d EXPERIMENTAL MODEL AND SUBJECT DETAILS B Bacterial strains, growth media, and culture conditions